新生大鼠HIBD远期神经功能和超微结构改变及早期运动训练对其影响的相关研究

发布时间：2018-06-02 18:38

本文选题：缺氧缺血性脑损伤 + 行为学　；参考：《南方医科大学》2012年硕士论文

【摘要】：研究背景新生儿缺氧缺血性脑损伤(Hypoxic-ischemic brain damage, HIBD)是指各种围产期窒息引起的部分或完全缺氧、脑血流减少或暂停而导致胎儿或新生儿脑损伤,临床出现一系列中枢神经系统异常的表现,是引起新生儿急性死亡和慢性神经系统损伤的主要原因之一,将遗留诸多后遗症,如脑性瘫痪、智力低下、癫痫及发育迟缓等。随着新生儿重症监护室的建立和医疗技术水平的提高,许多中、重度缺氧缺血性脑损伤患儿得以生存,但存活儿永久性脑功能障碍发病率仍较高。HIBD是一种弥漫性损伤,病变主要在大脑海马、皮层、纹状体、基底节、丘脑等部位,发病机制较复杂,是由多种机制综合作用所致的一系列生化连锁反应或称缺氧缺血性瀑布的结果。目前临床上对于新生儿HIBD治疗多采用对症处理、营养支持及早期干预等综合治疗,尚缺乏特效、经济的方法来治疗新生儿HIBD,因此深入探究HIBD机制和修复机制,寻找有效的防治措施,对于提高新生儿HIBD存活率,减少存活儿远期神经系统后遗症,提高我国人口质量均有重要意义。新生儿处在大脑发育关键期,而且新生儿HIBD具有非进行性、无持续性的缺血缺氧因素存在等特点,生后7d SD大鼠脑与新生儿脑发育相似,而且造成一过性脑缺氧缺血条件也与HIBD相似,因此,国内外以生后7d新生SD大鼠为研究对象,参照Rice方法,建立新生鼠HIBD动物模型,该模型被广泛应用于研究HIBD相关课题。在上述基础上,利用动物模型,深入探讨HIBD后的损伤机制,了解缺氧缺血对新生大鼠远期神经功能和超微结构的影响,探索合理、全面的判断预后和评估疗效的方法,进一步寻求有效、经济的干预措施,并探讨干预措施的修复机制。 HIBD是一种弥漫性损伤,由于HIBD致病因素的复杂性,加上婴幼儿脑发育的特殊性和可塑性,临床上要准确地评价HIBD的远期预后及干预疗效相当困难,许多患儿的预后与急性期损伤的严重程度并无一致的线性相关。而目前关于HIBD动物实验研究中,对于HIBD远期预后及干预措施疗效的评价多停留在行为学及上述两个脑区损伤后常规病理学改变的观察上,鲜有超微结构的研究。突触是神经元和效应细胞之间传递信息的功能部位,与行为学表现之间关系密切,也是脑发育可塑性的核心。因此,观察突触超微结构的改变不仅可以深入探究HIBD机理及修复机制,还可以了解其与行为学之间的相关性,从而有望对HIBD远期预后及干预疗效做出早期客观的评价。目前对于新生儿HIBD的治疗措施主要有高压氧、亚低温、神经营养药物及一般支持对症处理,高压氧治疗方法受医院条件设备限制、早期病情程度亦影响其应用,且治疗时间窗、疗程尚无统一定论等,而亚低温治疗方法不仅疗效不确定,而且应用范围较为局限,神经营养药物目前研究报道仅提示对疾病急性期有一定效果,且治疗费用高,影响其在临床上的应用。运动训练是指通过病人自身力量或治疗师的辅助操作或借助于器械所进行的主动或被动运动以改善局部或全身功能为目的的一种治疗方法。因其实施方便、费用低、治疗可长期坚持并可在家中实施,在临床上已应用于新生儿HIBD的治疗,取得一定疗效,但其具体机制尚未完全明确。一方面因为新生儿大脑正处在发育关键期,存在着较强可塑性,对损伤有一定自身修复能力,临床上很难判断患儿的进步是否与此干预治疗相关。另外,研究表明,适时适量的运动训练不仅能改善运动功能,而且可以提高人和动物的学习记忆能力。新生儿HIBD除运动功能障碍外,许多伴有智力低下、感觉统合失调等问题,早期运动训练是否也能改善患儿这些功能呢?是通过何种可能途径改善的呢? 据研究报道,适宜的运动训练可以引起中枢神经系统内cAMP、Ca2+等第二信使适量增加,从而诱导c-fos基因表达,控制下游靶基因的转录,从而合成新的蛋白质。在正常情况下,c-fos蛋白参与细胞生长、分化、信息传递、学习和记忆等生理过程,它对于编码记忆,形成大多数的长时程增强(Long-term potentiation, LTP)有着十分重要的作用,而LTP是神经元可塑性的反映。因此,有利于大脑的记忆功能,这为运动对学习记忆的促进作用在分子水平上提供一个证据。已有研究指出突触素(p38)是突触重建的重要标志,因为p38参与神经元发育和分化过程。发育中神经元p38的表达优先于突触的构成,即在突触结构形成之前就已经有p38存在和表达,随着神经元发育成熟、突触成熟,p38的表达也相应增加。p38还直接参与了突触的形成及调节与突触相关的其它突触蛋白的表达。因此该蛋白被广泛用于标记突触终末。基于以上理论,本研究采用Rice方法建立新生大鼠HIBD模型,观察大鼠远期行为学、病理学及超微结构的改变,探索HIBD机制,重点观察超微结构的改变及其意义,指导HIBD远期预后及疗效评估。之后早期给予运动训练干预,观察其对新生大鼠HIBD远期运动、学习记忆能力及感觉功能的疗效,并利用免疫组织化学方法检测c-fos、突触素的表达,来初步探索其疗效机制。具体研究内容包括以下两个部分：第一部分构建新生大鼠HIBD动物模型,观察远期行为学及超微结构改变目的建立新生大鼠缺氧缺血性脑损伤(Hypoxic-ischemic brain damage, HIBD)模型,观察远期行为学及超微结构改变,提供HIBD远期功能评价方法及指导临床治疗。方法 60只7日龄新生SD大鼠随机分为HIBD组和假手术组,每组30只。生后5周进行Morris水迷宫及感觉功能测试；取脑组织切片行尼氏染色计数神经元数目；取皮层、海马行透射电镜,观察突触结构、测量突触后致密区厚度及活性区长度,并与行为学结果进行相关分析。结果 Morris水迷宫测试中,HIBD组大鼠寻找平台潜伏期时间明显长于假手术组(P0.05);HIBD组大鼠穿越平台次数较假手术组少(P0.05)。HIBD组感觉运动功能测试结果明显差于假手术组。尼氏染色提示HIBD组神经元数目明显减少(P0.01)。电镜显示,HIBD组大鼠突触数量减少,突触后致密区厚度及活性区长度变薄变短。HIBD组大鼠海马突触后致密区厚度与Morris水迷宫寻找平台潜伏期时间呈负相关(r=-0.861,P=0.006),与三项感觉功能评估得分之和呈负相关性(r=-0.758,P=0.029)。结论缺氧缺血可致新生鼠远期神经元减少和超微结构损伤,造成远期行为学功能障碍,超微结构改变在一定程度上优于病理观察,有助于更好评估预后和评价疗效,基于未成熟脑突触的可塑性,在脑发育关键期指导临床治疗。第二部分早期运动训练对新生大鼠HlBD远期神经功能的影响目的探讨早期运动训练对新生大鼠缺氧缺血性脑损伤远期病理、超微结构、神经功能的影响。方法 90只7日龄SD大鼠随机分为运动组、对照组和假手术组。运动组和对照组建立HIBD模型,运动组于HIBD后1周开始每天给予抓握、旋转、行走、平衡等训练。4周后检测各组大鼠神经功能评分、空间学习记忆和皮层感觉功能、透射电镜观察突触和神经元超微结构,HE染色观察脑组织病理改变,尼氏染色计数神经元数目,并检测突触素、c-fos表达水平。结果运动组神经功能评分、空间学习记忆、皮层感觉功能均优于对照组,和假手术组间差异无统计学意义。透射电镜下对照组大鼠海马和皮层突触损伤严重,神经毡区突触减少,突触前膨大肿胀、轮廓不清晰,突触小泡溶解空泡形成,PSD变薄、薄厚不均,而运动组未见明显异常。HE染色提示对照组神经元数目明显减少,可见肿胀坏死细胞,运动组与假手术组比较未见明显异常,尼氏染色提示对照组神经元数目明显减少,与运动组及假手术组比较差异有统计学意义。运动组大鼠海马和皮层突触素、c-fos表达水平明显高于对照组(均P0.05)。结论早期运动训练可减少缺氧缺血后海马和皮层神经元死亡,增强突触可塑性,从而改善HIBD后远期神经功能。早期运动训练使突触素和c-fos在海马和皮层强表达可能是其改善的机制之一。
[Abstract]:Research background
Neonatal hypoxic ischemic brain damage (Hypoxic-ischemic brain damage, HIBD) refers to the partial or complete hypoxia caused by perinatal asphyxia. The decrease or suspension of cerebral blood flow leads to brain damage in the fetus or newborn. A series of abnormalities of the central nervous system appear in the clinic, which causes acute death of the newborn and the chronic nervous system damage. One of the main causes of injury will remain a number of sequelae, such as cerebral palsy, mental retardation, epilepsy and retardation. With the establishment of the neonatal intensive care unit and the improvement of the level of medical technology, many of the children with severe hypoxic ischemic brain damage can survive, but the incidence of permanent brain dysfunction is still higher in.HIBD. The disease is mainly in the hippocampus, cortex, striatum, basal ganglia, thalamus, and other parts of the brain. The pathogenesis is complicated. It is the result of a series of biochemical chain reactions or hypoxic ischemic falls, which are caused by a variety of mechanisms. At present, most of the new HIBD treatments are treated with symptomatic treatment, nutritional support and early stage. Comprehensive treatment, such as intervention, is still lack of special effects and economic methods for the treatment of neonatal HIBD. Therefore, it is of great significance to explore the mechanism of HIBD and repair mechanism and find effective measures to improve the survival rate of the newborn HIBD, reduce the sequelae of the long-term nervous system and improve the quality of the human mouth of our country.
The newborns are in the key stage of brain development, and the HIBD of the newborn is non progressive and has no persistent ischemic and anoxic factors. The brain of the 7d SD rats after birth is similar to the brain development of the newborn, and the condition of the hypoxic-ischemic cerebral ischemia is similar to that of the HIBD. Therefore, the newborn 7d newborn SD rat is the research object at home and abroad, with reference to Rice Methods the animal model of neonatal rat HIBD was established, and the model was widely used in the research of HIBD related subjects. On the basis of the above, the damage mechanism of HIBD was explored by animal model, and the effects of hypoxia ischemia on the long-term neural function and ultrastructure of neonatal rats were investigated. We should further seek effective and economic intervention measures and explore the repair mechanism of intervention measures.
HIBD is a diffuse injury. Due to the complexity of the pathogenic factors of HIBD and the specificity and plasticity of the brain development of infants, it is very difficult to accurately evaluate the long-term prognosis of HIBD and the effect of intervention. The prognosis of many children has no linear correlation with the severity of the acute phase. At present, the HIBD animal is real. In the study, the evaluation of the long-term prognosis of HIBD and the effect of intervention measures remained mostly in the observation of behavior and the routine pathological changes of the two brain regions, and there were few ultrastructural studies. The synapse is the functional part of the transmission of information between the neurons and the effector cells, which is closely related to the behavior and the development of the brain. Therefore, observing the changes in the synaptic ultrastructure can not only explore the mechanism of HIBD and the mechanism of repair, but also understand the correlation between the ultrastructure and the behavior. It is expected to make an early and objective evaluation of the long-term prognosis of HIBD and the effect of intervention.
At present, the treatment measures for neonatal HIBD mainly include hyperbaric oxygen, hypothermia, neurotrophic drugs and general support for symptomatic treatment. Hyperbaric oxygen therapy is restricted by hospital condition equipment, and the early condition also affects its application, and the treatment time window has no definite theory, and the mild hypothermia therapy not only has no definite effect, but the treatment of mild hypothermia is not effective, but the treatment of mild hypothermia is not definite, but And the application scope is limited. The present study of neurotrophic drugs only suggests that it has some effect on the acute phase of the disease, and the cost of treatment is high, which affects its clinical application. Exercise training refers to the improvement of the local or the whole by the auxiliary operation of the patient's own strength or therapist or by the active or passive motion of the apparatus. Physical function is a kind of therapeutic method. Because of its convenient implementation, low cost, the treatment can be persisted in the long term and can be implemented in the home. It has been applied to the treatment of HIBD in the newborn, and has achieved certain effect. But the specific mechanism is not completely clear. On the one hand, the brain is in the key stage of development, and there is a strong plasticity. It is difficult to judge whether the progress of the children is related to the intervention treatment in clinic. In addition, the study shows that appropriate exercise training can not only improve the exercise function, but also improve the learning and memory ability of the human and animal. In addition to the dysfunction of the movement of the newborn HIBD, many of them are accompanied by mental retardation. Problems such as maladjustment and so on, can early exercise training improve these functions of children?
According to the study, appropriate exercise training can cause the cAMP, Ca2+ and other two messenger to increase in the central nervous system, thus inducing c-fos gene expression, controlling the transcription of the target genes downstream, and synthesizing new proteins. Under normal conditions, c-fos protein participates in cell growth, differentiation, information transmission, learning and memory, and other physiological processes. Long-term potentiation (LTP) plays a very important role in coding memory, and LTP is a reflection of the plasticity of neurons. Therefore, it is beneficial to the memory function of the brain, which provides an evidence on the molecular level of the effect of exercise on learning and memory.
It has been pointed out that synaptophysin (p38) is an important sign of synaptic reconstruction, because p38 participates in the process of neuronal development and differentiation. The expression of p38 in the developing neuron is preceded by the composition of the synapse, that is, the presence and expression of p38 before the formation of the synapse structure, with the maturation of the neurons, the maturation of the synapses, and the corresponding increase of.P38 in the expression of p38. It also directly participates in synapse formation and regulates the expression of other synapses related to synapses. Therefore, the protein is widely used to mark synaptic terminals.
Based on the above theory, the Rice method was used to establish the HIBD model of neonatal rats, to observe the changes in the long term behavior, pathology and ultrastructure of rats, to explore the HIBD mechanism, to observe the changes and significance of the ultrastructure, to guide the long-term prognosis and the evaluation of the curative effect of HIBD. BD long term exercise, learning and memory ability and sensory function, and using immunohistochemical method to detect the expression of c-fos and synaptophysin to explore its therapeutic mechanism.
The specific research contents include the following two parts:
The first part is to construct the neonatal rat HIBD animal model, and to observe the long-term behavior and ultrastructural changes.
To establish the Hypoxic-ischemic brain damage (HIBD) model of neonatal rats, observe the long-term behavioral and ultrastructural changes, provide the method of evaluating the long-term function of HIBD and guide the clinical treatment.
Method
60 newborn SD rats at 7 days of age were randomly divided into HIBD group and sham operation group, with 30 rats in each group. The Morris water maze and sensory function test were carried out at 5 weeks after birth. The number of neurons was counted by Nissl staining in the brain tissue sections. The cortex and hippocampal transmission electron microscope were used to observe the synaptic structure, and the thickness of the dense area and the length of the active area after the contact were measured, and the behavior was measured and acted on the behavior. The results of the study were analyzed.
Result
In the Morris water maze test, the latency time of the HIBD group was significantly longer than that of the sham operation group (P0.05), and the number of HIBD rats crossing the platform was less than the sham operation group (P0.05).HIBD group was significantly worse than the sham operation group. Nissl staining suggested that the number of neurons in the HIBD group decreased significantly (P0.01). The electron microscope showed that the HIBD group was large. The number of rat synapses was reduced, the thickness of the post synaptic density and the length of the active region became thinner in the.HIBD group. The thickness of the postsynaptic density in the hippocampus of the hippocampus was negatively correlated with the latency time of the Morris water maze finding platform (r=-0.861, P=0.006), which was negatively correlated with the sum of three sensory evaluation scores (r=-0.758, P=0.029).
conclusion
Hypoxic-ischemic can cause long-term neuronal reduction and ultrastructural damage in neonatal rats, resulting in long-term behavioral dysfunction. Ultrastructural changes are superior to pathological observation to a certain extent. It is helpful to better evaluate the prognosis and evaluate the curative effect. Based on the plasticity of immature brain synapses, it guides clinical treatment at the critical stage of brain development.
The second part is the effect of early exercise training on the long-term neurological function of neonatal rats with HlBD.
objective
Objective to investigate the effects of early exercise training on long-term pathology, ultrastructure and neurological function in neonatal rats with hypoxic-ischemic brain damage.
Method
90 7 day old SD rats were randomly divided into exercise group, control group and sham operation group. HIBD model was established in the exercise group and the control group. The exercise group began to grasp, rotate, walk and balance every day after the 1 weeks of HIBD. After.4 weeks, the neural function score of each group was detected, the space learning memory and cortical sensory function were observed, and the synapse and the God were observed by transmission electron microscope. The pathological changes of brain tissue were observed by HE staining, Nissl staining was used to count the number of neurons, and synaptophysin and c-fos expression levels were detected.
Result
The neural function score, spatial learning and memory, cortical sensory function in the exercise group were better than the control group, and there was no statistical difference between the sham operation group and the sham operation group. Under transmission electron microscope, the hippocampus and cortical synapses in the control group were seriously damaged, the synapses in the nerve felt area decreased, the swelling of the synapse was not clear, the synaptic vesicle dissolved vacuoles, the PSD thinner, and the thinning of the synapse. There was no obvious abnormal.HE staining in the exercise group, which showed that the number of neurons in the control group decreased significantly, and the swelling and necrotic cells were visible. The number of neurons in the exercise group and the sham operation group was not obvious. The number of neurons in the control group decreased significantly, and the difference was statistically significant between the control group and the sham operation group. The expression levels of synaptophysin and c-fos in horses and cortex were significantly higher than those in control group (P0.05).
conclusion
Early exercise training can reduce the death of hippocampal and cortical neurons after hypoxia and ischemia, enhance synaptic plasticity, and improve the long-term neurological function after HIBD. Early exercise training may be one of the mechanisms for improving the expression of synaptophysin and c-fos in the hippocampus and cortex.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R722.1

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